Correction: A line-broadening free real-time 31P pure shift NMR method for phosphometabolomic analysis.

Correction for 'A line-broadening free real-time 31P pure shift NMR method for phosphometabolomic analysis' by Karl Kristjan Kaup et al., Analyst, 2021, 146, 5502-5507, DOI: 10.1039/D1AN01198G.

A line-broadening free real-time 31P pure shift NMR method for phosphometabolomic analysis.

Phosphometabolomics by 31P NMR can be challenging, since overlapping multiplets of homonuclear coupled phosphorus nuclei complicate spectral analysis. Pure shift NMR allows to simplify such spectra by collapsing multiplets into singlets, but most pure shift methods require substantially elongated measurement times or cause disturbing spectral line broadening. Herein, we combine established pure shift NMR and artefact suppression techniques to record 31P pure shift NMR spectra without penalties in measurement time or line width. Examples are demonstrated in resolution of a mixture of nucleotide triphosphates and a biological sample of 18O labelled ATP isotopomers.

Colon cancer cell differentiation by sodium butyrate modulates metabolic plasticity of Caco-2 cells via alteration of phosphotransfer network.

The ability of butyrate to promote differentiation of cancer cells has important implication for colorectal cancer (CRC) prevention and therapy. In this study, we examined the effect of sodium butyrate (NaBT) on the energy metabolism of colon adenocarcinoma Caco-2 cells coupled with their differentiation. NaBT increased the activity of alkaline phosphatase indicating differentiation of Caco-2 cells. Changes in the expression of pluripotency-associated markers OCT4, NANOG and SOX2 were characterized during the induced differentiation at mRNA level along with the measures that allowed distinguishing the expression of different transcript variants. The functional activity of mitochondria was studied by high-resolution respirometry. Glycolytic pathway and phosphotransfer network were analyzed using enzymatical assays. The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory capacity and caused rearrangement of the cellular phosphotransfer networks. The flexibility of phosphotransfer networks depended on the availability of glutamine, but not glucose in the cell growth medium. These changes were accompanied by suppressed cell proliferation and altered gene expression of the main pluripotency-associated transcription factors. This study supports the view that modulating cell metabolism through NaBT can be an effective strategy for treating CRC. Our data indicate a close relationship between the phosphotransfer performance and metabolic plasticity of CRC, which is associated with the cell differentiation state.

Metabolic and OXPHOS Activities Quantified by Temporal ex vivo Analysis Display Patient-Specific Metabolic Vulnerabilities in Human Breast Cancers.

Research on mitochondrial metabolism and respiration are rapidly developing areas, however, efficient and widely accepted methods for studying these in solid tumors are still missing. Here, we developed a new method without isotope tracing to quantitate time dependent mitochondrial citrate efflux in cell lines and human breast cancer samples. In addition, we studied ADP-activated respiration in both of the sample types using selective permeabilization and showed that metabolic activity and respiration are not equally linked. Three times lower amount of mitochondria in scarcely respiring MDA-MB-231 cells convert pyruvate and glutamate into citrate efflux at 20% higher rate than highly respiring MCF-7 mitochondria do. Surprisingly, analysis of 59 human breast cancers revealed the opposite in clinical samples as aggressive breast cancer subtypes, in comparison to less aggressive subtypes, presented with both higher mitochondrial citrate efflux and higher respiration rate. Additionally, comparison of substrate preference (pyruvate or glutamate) for both mitochondrial citrate efflux and respiration in triple negative breast cancers revealed probable causes for high glutamine dependence in this subtype and reasons why some of these tumors are able to overcome glutaminase inhibition. Our research concludes that the two widely used breast cancer cell lines fail to replicate mitochondrial function as seen in respective human samples. And finally, the easy method described here, where time dependent small molecule metabolism and ADP-activated respiration in solid human cancers are analyzed together, can increase success of translational research and ultimately benefit patients with cancer.

Mitochondrial Respiration in KRAS and BRAF Mutated Colorectal Tumors and Polyps.

This study aimed to characterize the ATP-synthesis by oxidative phosphorylation in colorectal cancer (CRC) and premalignant colon polyps in relation to molecular biomarkers KRAS and BRAF. This prospective study included 48 patients. Resected colorectal polyps and postoperative CRC tissue with adjacent normal tissue (control) were collected. Patients with polyps and CRC were divided into three molecular groups: KRAS mutated, BRAF mutated and KRAS/BRAF wild-type. Mitochondrial respiration in permeabilized tissue samples was observed using high resolution respirometry. ADP-activated respiration rate (Vmax) and an apparent affinity of mitochondria to ADP, which is related to mitochondrial outer membrane (MOM) permeability, were determined. Clear differences were present between molecular groups. KRAS mutated CRC group had lower Vmax values compared to wild-type; however, the Vmax value was higher than in the control group, while MOM permeability did not change. This suggests that KRAS mutation status might be involved in acquiring oxidative phenotype. KRAS mutated polyps had higher Vmax values and elevated MOM permeability as compared to the control. BRAF mutated CRC and polyps had reduced respiration and altered MOM permeability, indicating a glycolytic phenotype. To conclude, prognostic biomarkers KRAS and BRAF are likely related to the metabolic phenotype in CRC and polyps. Assessment of the tumor mitochondrial ATP synthesis could be a potential component of patient risk stratification.

Serum Amyloid Alpha Is Downregulated in Peripheral Tissues of Parkinson's Disease Patients.

We report the changed levels of serum amyloid alpha, an immunologically active protein, in Parkinson's disease (PD) patients' peripheral tissues. We have previously shown that Saa-1 and -2 (serum amyloid alpha-1,-2, genes) were among the top downregulated genes in PD patients' skin, using whole-genome RNA sequencing. In the current study, we characterized the gene and protein expression profiles of skin and blood samples from patients with confirmed PD diagnosis and age/sex matched controls. qRT-PCR analysis of PD skin demonstrated downregulation of Saa-1 and -2 genes in PD patients. However, the lowered amount of protein could not be visualized using immunohistochemistry, due to low quantity of SAA (Serum Amyloid Alpha, protein) in skin. Saa-1 and -2 expression levels in whole blood were below detection threshold based on RNA sequencing, however significantly lowered protein levels of SAA1/2 in PD patients' serum were shown with ELISA, implying that SAA is secreted into the blood. These results show that SAA is differentially expressed in the peripheral tissues of PD patients.

The complexity of mitochondrial outer membrane permeability and VDAC regulation by associated proteins.

Previous studies have shown that class II ß-tubulin plays a key role in the regulation of oxidative phosphorylation (OXPHOS) in some highly differentiated cells, but its role in malignant cells has remained unclear. To clarify these aspects, we compared the bioenergetic properties of HL-1 murine sarcoma cells, murine neuroblastoma cells (uN2a) and retinoic acid - differentiated N2a cells (dN2a). We examined the expression and possible co-localization of mitochondrial voltage dependent anion channel (VDAC) with hexokinase-2 (HK-2) and ßII-tubulin, the role of depolymerized ßII-tubuline and the effect of both proteins in the regulation of mitochondrial outer membrane (MOM) permeability. Our data demonstrate that neuroblastoma and sarcoma cells are prone to aerobic glycolysis, which is partially mediated by the presence of VDAC bound HK-2. Microtubule destabilizing (colchicine) and stabilizing (taxol) agents do not affect the MOM permeability for ADP in N2a and HL-1 cells. The obtained results show that ßII-tubulin does not regulate the MOM permeability for adenine nucleotides in these cells. HL-1 and NB cells display comparable rates of ADP-activated respiration. It was also found that differentiation enhances the involvement of OXPHOS in N2a cells due to the rise in their mitochondrial reserve capacity. Our data support the view that the alteration of mitochondrial affinity for ADNs is one of the characteristic features of cancer cells. It can be concluded that the binding sites for tubulin and hexokinase within the large intermembrane protein supercomplex Mitochondrial Interactosome, could be different between muscle and cancer cells.

Comparative analysis of the bioenergetics of human adenocarcinoma Caco-2 cell line and postoperative tissue samples from colorectal cancer patients.

The aim of this work was to explore the key bioenergetic properties for mitochondrial respiration in the widely-used Caco-2 cell line and in human colorectal cancer (HCC) postoperational tissue samples. Oxygraphy and metabolic control analysis (MCA) were applied to estimate the function of oxidative phosphorylation in cultured Caco-2 cells and HCC tissue samples. The mitochondria of Caco-2 cells and HCC tissues displayed larger functional activity of respiratory complex (C)II compared with CI, whereas in normal colon tissue an inverse pattern in the ratio of CI to CII activity was observed. MCA showed that the respiration in Caco-2 and HCC tissue cells is regulated by different parts of electron transport chain. In HCC tissues, this control is performed essentially at the level of respiratory chain complexes I-IV, whereas in Caco-2 cells at the level of CIV (cytochrome c oxidase) and the ATP synthasome. The differences we found in the regulation of respiratory chain activity and glycose index could represent an adaptive response to distinct growth conditions; this highlights the importance of proper validation of results obtained from in-vitro models before their extrapolation to the more complex in-vivo systems.

Mitochondrial Respiration in Human Colorectal and Breast Cancer Clinical Material Is Regulated Differently.

We conducted quantitative cellular respiration analysis on samples taken from human breast cancer (HBC) and human colorectal cancer (HCC) patients. Respiratory capacity is not lost as a result of tumor formation and even though, functionally, complex I in HCC was found to be suppressed, it was not evident on the protein level. Additionally, metabolic control analysis was used to quantify the role of components of mitochondrial interactosome. The main rate-controlling steps in HBC are complex IV and adenine nucleotide transporter, but in HCC, complexes I and III. Our kinetic measurements confirmed previous studies that respiratory chain complexes I and III in HBC and HCC can be assembled into supercomplexes with a possible partial addition from the complex IV pool. Therefore, the kinetic method can be a useful addition in studying supercomplexes in cell lines or human samples. In addition, when results from culture cells were compared to those from clinical samples, clear differences were present, but we also detected two different types of mitochondria within clinical HBC samples, possibly linked to two-compartment metabolism. Taken together, our data show that mitochondrial respiration and regulation of mitochondrial membrane permeability have substantial differences between these two cancer types when compared to each other to their adjacent healthy tissue or to respective cell cultures.

Changes in the mitochondrial function and in the efficiency of energy transfer pathways during cardiomyocyte aging.

The role of mitochondria in alterations that take place in the muscle cell during healthy aging is a matter of debate during recent years. Most of the studies in bioenergetics have a focus on the model of isolated mitochondria, while changes in the crosstalk between working myofibrils and mitochondria in senescent cardiomyocytes have been less studied. The aim of our research was to investigate the modifications in the highly regulated ATP production and energy transfer systems in heart cells in old rat cardiomyocytes. The results of our work demonstrated alterations in the diffusion restrictions of energy metabolites, manifested by changes in the apparent Michaelis-Menten constant of mitochondria to exogenous ADP. The creatine kinase (CK) phosphotransfer pathway efficiency declines significantly in senescence. The ability of creatine to stimulate OXPHOS as well as to increase the affinity of mitochondria for ADP is falling and the most critical decline is already in the 1-year group (middle-age model in rats). Also, a moderate decrease in the adenylate kinase phosphotransfer system was detected. The importance of glycolysis increases in senescence, while the hexokinase activity does not change during healthy aging. The main result of our study is that the decline in the heart muscle performance is not caused by the changes in the respiratory chain complexes activity but mainly by the decrease in the energy transfer efficiency, especially by the CK pathway.

Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes.

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding Km values were measured as 93.6±7.7 µM for CRC cells and 84.9±9.9 µM for nearby tissue; both these apparent Km (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34 µM).

An in situ study of bioenergetic properties of human colorectal cancer: the regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome.

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used. Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis-Menten constant (Km) for ADP and maximal respiratory rate (Vm) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (Km = 260 ± 55 µM) whereas the affinity of tumor mitochondria was significantly higher (Km = 126 ± 17 µM). But concurrently the Vm value of the tumor samples was 60-80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin ß-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides. The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase. Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed. Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.

Initiation and developmental dynamics of Wfs1 expression in the context of neural differentiation and ER stress in mouse forebrain.

Wolframin (Wfs1) is a membrane glycoprotein that resides in the endoplasmic reticulum (ER) and regulates cellular Ca(2+) homeostasis. In pancreas Wfs1 attenuates unfolded protein response (UPR) and protects cells from apoptosis. Loss of Wfs1 function results in Wolfram syndrome (OMIM 222300) characterized by early-onset diabetes mellitus, progressive optic atrophy, diabetes insipidus, deafness, and psychiatric disorders. Similarly, Wfs1-/- mice exhibit diabetes and increased basal anxiety. In the adult central nervous system Wfs1 is prominent in central extended amygdala, striatum and hippocampus, brain structures largely involved in behavioral adaptation of the organism. Here, we describe the initiation pattern of Wfs1 expression in mouse forebrain using mRNA in situ hybridization and compare it with Synaptophysin (Syp1), a gene encoding synaptic vesicle protein widely used as neuronal differentiation marker. We show that the expression of Wfs1 starts during late embryonic development in the dorsal striatum and amygdala, then expands broadly at birth, possessing several transitory regions during maturation. Syp1 expression precedes Wfs1 and it is remarkably upregulated during the period of Wfs1 expression initiation and maturation, suggesting relationship between neural activation and Wfs1 expression. Using in situ hybridization and quantitative real-time PCR we show that UPR-related genes (Grp78, Grp94, and Chop) display dynamic expression in the perinatal brain when Wfs1 is initiated and their expression pattern is not altered in the brain lacking functional Wfs1.